Project #3: IGF-Signal Transduction Pathway in Medulloblastoma A considerable line of evidence points at the IGF-I auto/paracrine system as an important component in the development and progression of brain tumors. Despite this, little attention has been paid to the role of IGF-I receptor (IGF-IR), and its signaling pathways, in primitive neuroectodermal tumors/ medulloblastomas (PNETs/MBs) that represent about 25% of all pediatric brain tumors. This research proposal is founded on the hypothesis that the IGF-IR system and the JCV T-antigen cooperate in the development and/or progression of medulloblastomas. Importance of this hypothesis is supported by several findings: (i) JC virus (JCV) infects greater than 80% of the human population; (ii) JCV T-antigen was found in human tumors including PNET/MBs; (iii) ectopic expression of JCV T-antigen transforms cells in culture and is tumorogenic in experimental animals; and (iv) JCV T-antigen, as well as, its murine counterpart - SV40 T-antigen, are not able to transform cells that do not possess functional IGF-IR. To further support our hypothesis, we have developed new evidence demonstrating overexpression of the major IGF-IR substrate, IRS-1, in both human and mouse medulloblastoma cell lines, and the activation of IGF-I system including constitutive phosphorylation of the IGF-IR protein in biopsies from patients with medulloblastoma. We have also found that both JCV-T-antigen and IGF-IR are necessary for medulloblastoma cell lines to survive and grow in anchorage-independent culture condition, and finally, we have demonstrated that JCV T-antigen and IRS-1 interact with each other. Three specific aims are proposed to directly test the hypothesis on the functional role for IGF-1 signaling pathway in the genesis of medulloblastoma; in the first aim mutational analysis of the IGF-IR will be utilized to determine whether functional interaction between the IGF-IR and JCV T-antigen contributes to malignant transformation in medulloblastomas; in the second aim JCV T-antigen positive and negative medulloblastoma cell lines will be employed to determine whether a unique set of IGF-IR pathways is involved in JCV T-antigen mediated transformation. Metabolic inhibitors in combination with mutational analysis of signaling molecules, including IRS-1 and PTEN phosphatase, will be employed to alter IGF-IR pathways and to test their importance in both T-antigen and non T-antigen mediated transformation; finally in the third aim dominant negative strategies against the IGF-IR and IRS-1 function will be tested in vivo. The results of these in vivo studies will allow us to evaluate whether uncoupling of the IGF-IR signaling pathway/s from its functional synergy with JCV T-antigen will eliminate medulloblastoma tumors from cerebellar tissues.